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. 2015 May 15;24(10):1205-22.
doi: 10.1089/scd.2014.0413. Epub 2015 Jan 26.

Human mesenchymal stroma/stem cells exchange membrane proteins and alter functionality during interaction with different tumor cell lines

Yuanyuan Yang  1 , Anna Otte, Ralf Hass
Affiliations

Human mesenchymal stroma/stem cells exchange membrane proteins and alter functionality during interaction with different tumor cell lines

Yuanyuan Yang et al. Stem Cells Dev. .

Abstract

To analyze effects of cellular interaction between human mesenchymal stroma/stem cells (MSC) and different cancer cells, direct co-cultures were performed and revealed significant growth stimulation of the tumor populations and a variety of protein exchanges. More than 90% of MCF-7 and primary human HBCEC699 breast cancer cells as well as NIH:OVCAR-3 ovarian adenocarcinoma cells acquired CD90 proteins during MSC co-culture, respectively. Furthermore, SK-OV-3 ovarian cancer cells progressively elevated CD105 and CD90 proteins in co-culture with MSC. Primary small cell hypercalcemic ovarian carcinoma cells (SCCOHT-1) demonstrated undetectable levels of CD73 and CD105; however, both proteins were significantly increased in the presence of MSC. This co-culture-mediated protein induction was also observed at transcriptional levels and changed functionality of SCCOHT-1 cells by an acquired capability to metabolize 5'cAMP. Moreover, exchange between tumor cells and MSC worked bidirectional, as undetectable expression of epithelial cell adhesion molecule (EpCAM) in MSC significantly increased after co-culture with SK-OV-3 or NIH:OVCAR-3 cells. In addition, a small population of chimeric/hybrid cells appeared in each MSC/tumor cell co-culture by spontaneous cell fusion. Immune fluorescence demonstrated nanotube structures and exosomes between MSC and tumor cells, whereas cytochalasin-D partially abolished the intercellular protein transfer. More detailed functional analysis of FACS-separated MSC and NIH:OVCAR-3 cells after co-culture revealed the acquisition of epithelial cell-specific properties by MSC, including increased gene expression for cytokeratins and epithelial-like differentiation factors. Vice versa, a variety of transcriptional regulatory genes were down-modulated in NIH:OVCAR-3 cells after co-culture with MSC. Together, these mutual cellular interactions contributed to functional alterations in MSC and tumor cells.

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Figures

<b>FIG. 1.</b>
FIG. 1.
MCF-7cherry cells were incubated with three different human primary MSC populations (MSC240113GFP P2, MSC280313GFP P3, and MSC131113GFP P3) in three separate co-cultures at a ratio of 20% MCF-7/80% MSC with 500 cells/cm2 till 9 days. (A) Cell cycle analysis was performed in the co-cultures, and the ratio of the two populations (MCF-7cherry and MSCGFP) was quantified with the corresponding G1 peaks. Data represent the mean±SD from the three separate co-cultures. (B) The percentage of CD90 expression in each population of the co-culture was quantified by flow cytometry. Data represent the mean±SD from the three separate co-cultures. (C) Cell cycle analysis by flow cytometry and CD90 expression demonstrated little, if any, detection in MCF-7cherry and 97.4% in MSCGFP mono-cultures (upper panels), which were compared with an 8 day co-culture (middle panel). After separation of the co-cultured cells into cherry (red fluorescence) and GFP (green fluorescence) populations by sorting via FACS, subsequent flow cytometric analysis for CD90 and cell cycle revealed 91.3% CD90-positive MCF-7 cells. MSC, mesenchymal stroma/stem cells.
<b>FIG. 2.</b>
FIG. 2.
Co-culture of primary MSC with human primary breast cancer epithelial cells at a ratio of 40% HBCEC 699/60% MSC with 500 cells/cm2 till 10 days. (A) Detection and analysis of CD90 expression in mono-cultures of MSC280313 P5 and HBCEC 699. (B) Quantification of the percentage of MSC280313GFP P5 and HBCEC 699cherry and the formation of yellow chimeric/hybrid cells by flow cytometric analysis during a 10 day co-culture. (C) Quantification of the percentage of CD90-positive HBCEC 699 acquired during a 10 day co-culture with MSC. HBCEC, human breast cancer-derived epithelial cells.
<b>FIG. 3.</b>
FIG. 3.
Co-culture of primary MSC with human ovarian cancer cells at an initial ratio of 40% NIH:OVCAR-3/60% MSC with 2,000 cells/cm2 till 7 days. (A) Cell counting of MSC131113GFP P4 and NIH:OVCAR-3cherry and yellow chimeric/hybrid cells using a fluorescence microscope (Olympus IX50) with a FITC/TRIC fluorescence dual band filter and calculation of the population percentage during a 7 day co-culture. (B) Quantification of the percentage of MSC131113GFP P4 and NIH:OVCAR-3cherry and the formation of yellow chimeric/hybrid cells by flow cytometric analysis during a 7 day co-culture. (C) Quantification of the percentage of CD90-positive NIH:OVCAR-3 cells acquired during a 7 day co-culture with MSC. (D) Detection and analysis of CD90 expression in mono-cultures of MSC and NIH:OVCAR-3 cells.
<b>FIG. 3.</b>
FIG. 3.
Co-culture of primary MSC with human ovarian cancer cells at an initial ratio of 40% NIH:OVCAR-3/60% MSC with 2,000 cells/cm2 till 7 days. (A) Cell counting of MSC131113GFP P4 and NIH:OVCAR-3cherry and yellow chimeric/hybrid cells using a fluorescence microscope (Olympus IX50) with a FITC/TRIC fluorescence dual band filter and calculation of the population percentage during a 7 day co-culture. (B) Quantification of the percentage of MSC131113GFP P4 and NIH:OVCAR-3cherry and the formation of yellow chimeric/hybrid cells by flow cytometric analysis during a 7 day co-culture. (C) Quantification of the percentage of CD90-positive NIH:OVCAR-3 cells acquired during a 7 day co-culture with MSC. (D) Detection and analysis of CD90 expression in mono-cultures of MSC and NIH:OVCAR-3 cells.
<b>FIG. 4.</b>
FIG. 4.
Co-culture of primary MSC (MSC101213 P5 and MSC 131113 P4) with human ovarian adenocarcinoma cells at an initial ratio of 40% SK-OV-3/60% MSC with 2,000 cells/cm2 till 7 days. (A) Detection and analysis of CD90 and CD105 expression in mono-cultures of SK-OV-3 cells and MSC. (B) Quantification of the percentage of CD105-positive SK-OV-3 cells acquired during a 7 day co-culture with MSC101213. (C) Quantification of the percentage of CD90-positive SK-OV-3 cells acquired during a 7 day co-culture with MSC101213. (D) Cell counting of MSC131113GFP P4 and SK-OV-3cherry and yellow chimeric/hybrid cells using a fluorescence microscope (Olympus IX50) with an FITC/TRIC fluorescence dual band filter and calculation of the population percentage during a 7 day co-culture. (E) Quantification of the percentage of MSC131113GFP P4 and SK-OV-3cherry and the formation of yellow chimeric/hybrid cells by flow cytometric analysis during a 7 day co-culture.
<b>FIG. 4.</b>
FIG. 4.
Co-culture of primary MSC (MSC101213 P5 and MSC 131113 P4) with human ovarian adenocarcinoma cells at an initial ratio of 40% SK-OV-3/60% MSC with 2,000 cells/cm2 till 7 days. (A) Detection and analysis of CD90 and CD105 expression in mono-cultures of SK-OV-3 cells and MSC. (B) Quantification of the percentage of CD105-positive SK-OV-3 cells acquired during a 7 day co-culture with MSC101213. (C) Quantification of the percentage of CD90-positive SK-OV-3 cells acquired during a 7 day co-culture with MSC101213. (D) Cell counting of MSC131113GFP P4 and SK-OV-3cherry and yellow chimeric/hybrid cells using a fluorescence microscope (Olympus IX50) with an FITC/TRIC fluorescence dual band filter and calculation of the population percentage during a 7 day co-culture. (E) Quantification of the percentage of MSC131113GFP P4 and SK-OV-3cherry and the formation of yellow chimeric/hybrid cells by flow cytometric analysis during a 7 day co-culture.
<b>FIG. 5.</b>
FIG. 5.
Co-culture of primary MSC with human small cell ovarian carcinoma cells hypercalcemic type at an initial ratio of 40% SCCOHT-1/60% MSC with 2,000 cells/cm2 till 7 days. (A) Detection and analysis of CD73 and CD105 expression in mono-cultures of SCCOHT-1 cells and MSC. (B) Quantification of the percentage of CD73-positive SCCOHT-1 cells acquired during a 7 day co-culture with MSC101213. (C) Quantification of the percentage of CD105-positive SCCOHT-1 cells acquired during a 7 day co-culture with MSC101213. (D) Quantification of acquired 5′ nucleotidase enzymatic activity by liquid chromatography/tandem mass spectrometry analysis of 5′AMP and adenosine. Data represent the mean±SD of three independent experiments. A statistical analysis between the mono-culture and the corresponding population in co-culture was conducted by unpaired Student's t-test (**P< 0.01). (E) SCCOHT-1 cells previously co-cultured with MSC were separated by fluorescence-activated cell sorting (FACS), and sorted SCCOHT-1 cells were analyzed for purity by a GFP flow cytometry analysis. (F) RT-PCR of CD73 and CD105 transcripts (upper panel) and CD90 transcripts (lower panel) was performed in SCCOHT-1cherry and SK-OV-3cherry mono-cultures as compared with a 7 day co-culture with MSCGFP and subsequent separation by fluorescence-activated cell sorting. (G) RT-PCR of transcripts in MSCGFP and HMEC (P13) mono-cultures were compared with a 7 day co-culture and subsequent separation by fluorescence-activated cell sorting. Unaltered β-actin expression was used as a control. HMEC, human mammary epithelial cells; RT-PCR, reverse transcription PCR.
<b>FIG. 5.</b>
FIG. 5.
Co-culture of primary MSC with human small cell ovarian carcinoma cells hypercalcemic type at an initial ratio of 40% SCCOHT-1/60% MSC with 2,000 cells/cm2 till 7 days. (A) Detection and analysis of CD73 and CD105 expression in mono-cultures of SCCOHT-1 cells and MSC. (B) Quantification of the percentage of CD73-positive SCCOHT-1 cells acquired during a 7 day co-culture with MSC101213. (C) Quantification of the percentage of CD105-positive SCCOHT-1 cells acquired during a 7 day co-culture with MSC101213. (D) Quantification of acquired 5′ nucleotidase enzymatic activity by liquid chromatography/tandem mass spectrometry analysis of 5′AMP and adenosine. Data represent the mean±SD of three independent experiments. A statistical analysis between the mono-culture and the corresponding population in co-culture was conducted by unpaired Student's t-test (**P< 0.01). (E) SCCOHT-1 cells previously co-cultured with MSC were separated by fluorescence-activated cell sorting (FACS), and sorted SCCOHT-1 cells were analyzed for purity by a GFP flow cytometry analysis. (F) RT-PCR of CD73 and CD105 transcripts (upper panel) and CD90 transcripts (lower panel) was performed in SCCOHT-1cherry and SK-OV-3cherry mono-cultures as compared with a 7 day co-culture with MSCGFP and subsequent separation by fluorescence-activated cell sorting. (G) RT-PCR of transcripts in MSCGFP and HMEC (P13) mono-cultures were compared with a 7 day co-culture and subsequent separation by fluorescence-activated cell sorting. Unaltered β-actin expression was used as a control. HMEC, human mammary epithelial cells; RT-PCR, reverse transcription PCR.
<b>FIG. 6.</b>
FIG. 6.
Co-culture of primary MSC with different human ovarian carcinoma cells (SK-OV-3, NIH:OVCAR-3) at an initial ratio of 40% ovarian carcinoma cells/60% MSC with 2,000 cells/cm2 till 7 days. (A) Detection and analysis of steady-state CD326 (EpCAM) expression in mono-cultures of MSC101213, SK-OV-3, and NIH:OVCAR-3 cells, respectively. (B) Quantification of the percentage of EpCAM-positive MSC acquired during a 7 day co-culture with SK-OV-3 cells. (C) Quantification of the percentage of EpCAM-positive MSC acquired during a 7 day co-culture with NIH:OVCAR-3 cells. EpCAM, epithelial cell adhesion molecule.
<b>FIG. 7.</b>
FIG. 7.
(A) The morphology during co-culture of MSC180314GFP P3 and MCF-7cherry cells demonstrated various cellular interactions with the extension of nanotubes (yellow arrows), formation of exosomes (white arrows), and overlapping membranes. Scale bars represent 25 μm. (B) Co-culture of MSCGFP with different tumor cell lines demonstrated the formation of yellow chimeric/hybrid cells by simultaneous expression of cherry protein and eGPF (white arrows). Scale bars represent 100 μm. (C) Quantification of acquired CD90 by MCF-7, SK-OV-3, and NIH:OVCAR-3 cells after a 3 and 5 day co-culture with primary MSC180314 P3, respectively, and effect of 50 nM cytochalasin D in these co-cultures. (D) Quantification of acquired CD105 by SK-OV-3 cells within 3 days of co-culture with primary MSC100314 P3 (upper panel) and effect of 50 nM cytochalasin D in these co-cultures (lower panel). (E) Effect of cytochalasin D on the constitutive expression of CD90 and CD105 in 3 and 5 days cultured MSC270114 P2. (F) Flow cytometric analysis of double FACS-separated cells from a 7 day MSC180314GFP P6 and NIH:OVCAR-3cherry co-culture (left panels) and microarray analysis of the separated populations revealed selected prominent changes in gene expression of co-cultured MSCGFP (upper right panel) and co-cultured NIH:OVCAR-3cherry cells (lower right panel). Color images available online at www.liebertpub.com/scd
<b>FIG. 7.</b>
FIG. 7.
(A) The morphology during co-culture of MSC180314GFP P3 and MCF-7cherry cells demonstrated various cellular interactions with the extension of nanotubes (yellow arrows), formation of exosomes (white arrows), and overlapping membranes. Scale bars represent 25 μm. (B) Co-culture of MSCGFP with different tumor cell lines demonstrated the formation of yellow chimeric/hybrid cells by simultaneous expression of cherry protein and eGPF (white arrows). Scale bars represent 100 μm. (C) Quantification of acquired CD90 by MCF-7, SK-OV-3, and NIH:OVCAR-3 cells after a 3 and 5 day co-culture with primary MSC180314 P3, respectively, and effect of 50 nM cytochalasin D in these co-cultures. (D) Quantification of acquired CD105 by SK-OV-3 cells within 3 days of co-culture with primary MSC100314 P3 (upper panel) and effect of 50 nM cytochalasin D in these co-cultures (lower panel). (E) Effect of cytochalasin D on the constitutive expression of CD90 and CD105 in 3 and 5 days cultured MSC270114 P2. (F) Flow cytometric analysis of double FACS-separated cells from a 7 day MSC180314GFP P6 and NIH:OVCAR-3cherry co-culture (left panels) and microarray analysis of the separated populations revealed selected prominent changes in gene expression of co-cultured MSCGFP (upper right panel) and co-cultured NIH:OVCAR-3cherry cells (lower right panel). Color images available online at www.liebertpub.com/scd
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References

    1. Caplan AI. (1991). Mesenchymal stem cells. J Orthop Res 9:641–650 - PubMed
    1. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S. and Marshak DR. (1999). Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147 - PubMed
    1. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D. and Horwitz E. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8:315–317 - PubMed
    1. Simmons PJ. and Torok-Storb B. (1991). Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood 78:55–62 - PubMed
    1. Honczarenko M, Le Y, Swierkowski M, Ghiran I, Glodek AM. and Silberstein LE. (2006). Human bone marrow stromal cells express a distinct set of biologically functional chemokine receptors. Stem Cells 24:1030–1041 - PubMed

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